Studies in oxidative phosphorylation
Trialkyl tin compounds have been shown to be potent inhibitors of oxidative phosphorylation, oligomycin sensitive ATPase activity and other ATP-dependent reactions of beef heart mitochondria. Unlike oligomycin inhibition of these reactions, trialkyl tin inhibition is reversible by dithiols such as 2,3-dimercaptopropanol. The OS-ATPase activity was found to be 6-10 times more sensitive than oxidative phosphorylation to inhibition by trialkyl tins. This differential sensitivity to trialkyl tin inhibition is discussed in relation to the current theories on energy coupling. Binding studies with [3H]-DBCT have indicated the presence of two types of binding sites designated high affinity (kD ~ 0.3 uM) and low affinity (KD ~ 30 uM) binding sites. The concentration of the high affinity binding site is ~ 2.0 nmol/mg protein in submitochondrial particles and saturation of these sites correlated with the inhibition of the oligomycin sensitive ATPase activity. Extraction and isolation experiments have shown that DBCT binds to a small lipophilic, non-protein molecule. Dihydrolipoic acid has been shown to drive ATP synthesis by acting as a NAD-linked substrate. Fatty acids, oleoyl-phosphate, oleoyl-lipoate and other lipids were found to inhibit succinate driven ATP synthesis and other energy-linked reactions in mitochondria and submitochondrial particles. In addition, a new method for the measurement of nanomoles amount of lipoic acid and lipoamide is reported. The method involves the cyclic reduction of 5,5-dithiobis (2-nitrobenzoic acid) by NADH via a system containing lipoamide dehydrogenase and lipoic acid (or lipoamide).